Molecular Basis

of Inheritance
The DNA
➢ Over the years after Mendel, the nature of the genetic material was investigated. It resulted in the
realisation that DNA is the genetic material in majority of organisms.
➢ Deoxyribonucleic Acid (DNA) and Ribonucleic Acid (RNA) are the two types of nucleic acid
found in living systems.
➢ Nucleic acids are polymers of nucleotides (or a pair of nucleotide called base pairs).
➢ Bacteriophage Φ ×174 has 5386 nucleotides, bacteriophage λ(lambda) has 48502 bp, E. coli
has 4.6 × 106 bp and haploid content of human DNA has 3.3×109 bp.
➢ DNA acts as a genetic material in most organisms, whereas RNA acts as a genetic material in
some viruses.

Structure of Polynucleotide Chain

(i) A nucleotide has three parts, i.e. a nitrogenous base, a pentose sugar (deoxyribose in DNA and
ribose in RNA) and a phosphate group.
(ii) Nitrogenous bases can be purines, i.e. adenine and guanine or pyrimidines, i.e. cytosine, uracil
and thymine.

(iii) Cytosine is common for both DNA and RNA and thymine is present in DNA. Uracil is present
in RNA at the place of thymine.
(iv) A nitrogenous base is linked to the first carbon of pentose sugar through N-glycosidic linkage to
form a nucleoside, e.g. adenosine, guanosine, etc.

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 (v) When a phosphate group is linked to 5'-OH of a nucleoside on the fifth carbon of pentose sugar
through phosphoester linkage, a corresponding nucleotide is formed.
(vi) Two nucleotides are linked through 3'→ 5'phosphodiester linkage to form a dinucleotide.
(vii) Several nucleotides can be joined to form a polynucleotide chain. In this polynucleotide chain, at
one end free phosphate group is present which is known as 5' end, while at one end free OH group is
present, which is known as 3' end.
viii) The backbone in a polynucleotide chain is formed of sugar and phosphates.
(ix) The nitrogenous bases linked to sugar moiety project from the backbone.

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 Discoveries Related to Structure of DNA
(i) Friedrich Miescher (1869), first identified DNA as an acidic substance present in the
nucleus and named it as 'nuclein'. It was later named as nucleic acid by Altman (1899).
(ii) Levene (1910) found that DNA contains phosphoric acid as well as deoxyribose sugar. He
also found that four types of nucleotides are present in DNA.
(iii) Erwin Chargaff proposed that for a double-stranded DNA, the ratio between adenine (A),
thymine (T) and guanine (G), cytosine (C) are constant and equals to one.
A+T/ C+G =1
(iv) James Watson and Francis Crick, in 1953 proposed a very simple double helix model for
the structure of DNA based on X-ray diffraction data, produced by Maurice Wilkins and
Rosalind Franklin in the same year.

Salient Features of Double Helix Structure of DNA

(i) DNA is a long polymer of deoxyribonucleotides. It is made up of two polynucleotide chains,
where the backbone is constituted by sugar-phosphate and the bases project inside. So,
nitrogenous bases can be considered as steps of the coiled helix.
(ii) The two chains have anti-parallel polarity, i.e. 5'-3' for one, 3'→ 5' for another.
(iii) A base pair is formed by the pairing of nitrogenous bases in two strands through hydrogen
bonds (H-bonds). Adenine forms two hydrogen bonds with thymine on opposite strand.
Guanine bonds with cytosine by three H-bonds. Due to this, purine always comes opposite
to a pyrimidine. This forms a uniform distance between the two strands of the helix.
(iv) The two chains are coiled in a right-handed fashion. The pitch of the helix is 3.4 nm and
there are roughly 10 bp in each turn. Due to this, the distance between a base pair in a helix
is about 0.34 nm.
(v) The plane of one base pair stacks over the other in double helix. This confers stability to the
helical structure in addition to H-bonds.
(vi) Diameter of DNA double helix is 20 Å.
(vii) The length of a DNA double helix is about 2.2 metres in humans. (6.6×109 bp x 0.34 x10-9
m/bp). Therefore, it needs special packaging in a cell.

Packaging of DNA Helix

(i) In prokaryotic cells (which do not have a defined nucleus), such as E. coli, DNA being
negatively charged is held with some proteins that have positive charge in a region called as
nucleoid. The DNA in nucleoid is organised in large loops held by proteins. The length of
DNA in E. coli is around 1.36 mm.
(ii) In eukaryotes, there is a set of positively charged proteins called histones that are rich in
basic amino acid residues, lysines and arginines (both positive). The 5 types of histone
proteins are H1, H2 A, H2B, H3 and H4. These are organised to form a unit of 8 molecules
called histone octamer or nu-body.
In each octamer, 2 molecules of each H2A, H2B, H3 and H4 are present. The negatively
charged DNA is wrapped around the positively charged histone octamer to form a structure

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 called nucleosome. Two nucleosomes are connected to each other with the help of linker
DNA, on which H1 histone is present, So, H1, is also known as linker histone.

(iii) A typical nucleosome contains 200 bp of DNA helix. Nucleosomes are the repeating unit of a
structure called chromatin (i.e. thread-like stained structure) in the nucleus. Under electron
microscope, the nucleosomes in chromatin can be seen as beads-on-string.
(iv) The packaging of chromatin at higher level requires additional set of proteins, which are
collectively called Non-Histone Chromosomal (NHC) proteins.
(v) In a nucleus, some regions of chromatin are loosely packed (stains light) and are called
euchromatin (transcriptionally active chromatin). While in some regions, chromatin is
densely packed (stains dark) and are called as heterochromatin (transcriptionally
inactive chromatin).

The Search for Genetic Material

Transforming Principle
➢ Frederick Griffith (1928) carried out a series of experiments with Streptococcus pneumoniae
(bacterium causing pneumonia).
➢ According to him, when the bacteria are grown on a culture plate, some produce smooth shiny
colonies (S), while others produce rough (R) colonies.
➢ This is because the S-strain bacteria have a mucous (polysaccharide) coat, while R-strain does
not. So, S-strain was infectious, while R-strain was non-infectious.
➢ Griffith infected two groups of mice with S-strain and R-strain, respectively. Mice infected with
S-strain (virulent) died from pneumonia, but mice infected with R-straindid not develop
pneumonia.

➢ In an another set of experiment, Griffith killed bacteria by heating. He observed that on injecting
heat-killed S-strain bacteria, the mice remained alive. On injecting mixture of heat-killed S and
live R bacteria, the mice died.

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 ➢ He recovered both living S-bacteria and R-bacteria from dead mice and concluded that the
'R-strain bacteria' had been transformed by the heat-killed S-strain bacteria.
➢ Some transforming material had transferred from heat-killed S-strain to R-strain which enabled
the R-strain to synthesise a smooth polysaccharide coat in and became virulent.
➢ Later he concluded that this must had happen due to the transfer of the genetic material.
However, biochemical nature of genetic material was not defined from his experiments.

Biochemical Nature of Transforming Principle

➢ Oswald Avery, Colin MacLeod and Maclyn McCarty (1933-44), worked to determine the
biochemical nature of transforming principle in Griffith's experiment.
➢ They purified biochemicals such as proteins, RNA and DNA, etc., from heat-killed S-cells and
discovered that DNA alone from S-bacteria caused the transformation of R-bacteria.
➢ They also discovered that protease, i.e. protein digesting enzyme and RNases, i.e. RNA-
digesting enzymes did not affect transformation.
➢ Digestion of experimental material with DNase did inhibit transformation, indicating that
DNA caused transformation. Since, DNase digested DNA, transformation could not occur from
R-strain to S-strain. (v) They concluded that DNA is the hereditary material. But, still all the
biologists were not convinced.

The Genetic Material is DNA

➢ Alfred Hershey and Martha Chase (1952) gave unequivocal proof that DNA is the genetic
material.
➢ In their experiments, bacteriophages, i.e. viruses that infect bacteria, were used. Head of
bacteriophage is made up of protein and inside this protein coat DNA is present as genetic
material.
➢ They grew some viruses on a medium that contained radioactive phosphorus and some others on
sulphur containing radioactive medium. 32P was used as radioactive phosphorus, while 35S
was used as radioactive sulphur.
➢ Viruses grown in the presence of radioactive phosphorus contained radioactive DNA, but
not radioactive protein because DNA contains phosphorus, but protein does not. In the
same way, viruses grown on radioactive sulphur contained radioactive protein, but not
radioactive DNA because DNA does not contain sulphur while protein does.
➢ Radioactive phages were then allowed to attach to E. coli bacteria. As the infection proceeded,
viral coats were removed from the bacteria by agitating them in a blender. Following this,
the virus particles were separated from the bacteria by spinning them in a centrifuge.

➢ Bacteria which were infected with viruses containing radioactive DNA became radioactive
and 32P was detected in their cells, indicating that DNA was the material that passed from
the virus to the bacteria.
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 ➢ Bacteria that were infected with viruses containing radioactive proteins do not shown
radioactivity. 35S was not detected in the cells, while 35S was present in supernatant. This
indicated that the proteins did not enter the bacteria from viruses. Hence, it was proved that DNA
is a genetic material that is passed from virus to bacteria.

Properties of Genetic Material (DNA Versus RNA)

(i) Hershey-Chase experiment established that DNA is the genetic material.
(ii) In some viruses, RNA was also reported as genetic material, e.g. tobacco mosaic viruses,
QB bacteriophage, etc.
(iii) Characteristics of a Genetic Material
(a) It should be able to replicate.
(b) It should be chemically and structurally stable.
(c) It should provide scope for slow changes (mutation) that are required for evolution.
(d) It should be able to express itself in the form of 'Mendelian characters'.
➢ According to the above mentioned rules, both the nucleic acids (DNA and RNA) have the ability
to direct duplications, because of the rule of base pairing and complementarity.
➢ Stability can be explained in DNA by the fact that two strands if separated by heating, come
together in appropriate conditions and show complementarity.
➢ Presence of thymine also confers additional stability to DNA.

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 ➢ RNA is not stable as DNA. The 2'-OH group present at every nucleotide in RNA is a reactive
group and makes RNA labile and easily degradable, hence it is reactive. RNA is also catalytic in
nature. The catalytic RNA is called ribozyme.
➢ Hence, DNA is chemically less reactive and structurally more stable as compared to RNA.
Among the two nucleic acids, the DNA is a predominant genetic material.
➢ Both RNA and DNA are able to mutate. Viruses have RNA genome and shorter lifespan thus,
they mutate and evolve faster. So, faster mutation makes RNA more unstable.
➢ DNA is dependent on RNA for protein synthesis, while RNA can directly code for it. The protein
synthesising machinery has evolved around RNA.
➢ This concluded that the DNA being more stable is suitable for storage of genetic information,
while for the transmission of genetic information, RNA is suitable.

RNA World
➢ RNA was the first genetic material to be discovered. There are evidences to prove that
essential life processes, such as metabolism, translation, splicing, etc., have evolved around
RNA.
➢ There are some important biochemical reactions in living systems that are catalysed by RNA
catalysts and not by protein enzymes.
➢ DNA has evolved from RNA with chemical modifications that made it more stable, because
RNA being a catalyst was reactive and hence, unstable.

Types of RNA
There are following three types of RNAs
(i) mRNA (messenger RNA) provides the template for transcription.
(ii) tRNA (transfer RNA) brings amino acids and reads the genetic code.
(iii) rRNA (ribosomal RNA) plays structural and catalytic role during translation.
All the three RNAs are needed to synthesise a protein in a cell.

Central Dogma
Francis Crick proposed the central dogma in molecular biology, which states that genetic
information flows from DNA to RNA and then to proteins

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 ➢ In some viruses, the flow of information is in reverse direction, i.e. from RNA to DNA. This
process is known as reverse transcription. An enzyme is very important for this process known
as reverse transcriptase.

Replication
➢ The scheme for replication of DNA is termed as semi-conservative DNA replication. It was
proposed by Watson and Crick (1953).
➢ According to it,
(i) The two strands would separate and act as a template for the synthesis of new complementary
strands.
(ii) After replication, each DNA molecule would have one parental and one newly
synthesised strand.
(iii) The basis of semi-conservative method of replication is complementarity between
nitrogenous bases.

The Experimental Proof

Experimental proof that DNA replicates semi- conservatively, came first from E. coli and later
from higher organisms, such as plants and human
➢ Matthew Meselson and Franklin Stahl performed the following experiments to prove semi-
conservative mode of DNA replication in 1958.
The steps of this experiment are as follows
(i) E. coli was grown on a medium containing 15NH4Cl as the only nitrogen source for many
generations. 15N (heavy isotope of nitrogen) got incorporated into newly synthesised DNA
(and other nitrogen containing compounds). This heavy DNA molecule could be
distinguished from the normal DNA by centrifugation in a cesium chloride (CsCl) density
gradient.
(ii) They then transferred the cells into a medium with normal 14NH4Cl and took samples at
various definite intervals. As the cells multiplied and extracted, the DNA remained as double-
stranded helices. DNA samples were separated independently on CsCl gradients to measure
DNA densities.
(iii) The DNA that was extracted from the culture after the first generation (after 20 min), i.e. after
the transfer from 15N to 14N medium, had a hybrid or intermediate density. It means one
strand of DNA had 15N, while another strand had 14N. DNA extracted from the culture after
second generation (after 40 min) was composed of equal amount of hybrid DNA and light
(14N) DNA, i.e. one DNA molecule was of 15N 14N, while another DNA molecule was of 14N
14
N. So, it explains semi-conservative method of DNA replication.
(iv) Very similar experiments were carried out by Taylor and colleagues on Vicia faba (faba
beans) using radioactive thymidine and the same results were obtained as in earlier
experiments, i.e. DNA replicates semi-conservatively.

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 Machinery and Enzymes of DNA Replication
(i) DNA replication takes places in the S-phase of cell cycle.
(ii) It requires a set of catalysts or enzymes. The main enzyme is DNA-dependent DNA
polymerase, since it uses a DNA template to catalyse the polymerisation of
deoxynucleotides. The average rate of polymerisation by these enzymes is approximately
2000 bp/second in E. coli.
(iii) These polymerases has to catalyse the reaction with high degree of accuracy because any
mistake during replication would result into mutations.
(iv) DNA polymerisation is an energy demanding process, so deoxyribonucleoside
triphosphates serve dual purposes, i.e. act as substrates and provide energy for
polymerisation reaction (due to the presence of two terminal, high-energy phosphates).
(v) Many enzymes are also required in addition to DNA-dependent DNA polymerase, such as
helicase, topoisomerase, SSB, ligase, etc.
(vi) Both the strands of DNA get separated by the activity of enzyme helicase. So, it causes
unwinding of DNA.
(vii) Topoisomerase enzyme removes tension from uncoiled DNA strand.
(viii) SSB protein stabilises uncoiled DNA strand so that it cannot coil again.
(ix) (a) Replication in DNA strand occurs within a small opening of the DNA helix, known as
replication fork. It is a Y-shaped structure formed due to uncoiling of DNA.

(b) DNA polymerase cannot initiate the process of replication on their own, so it needs a
primer to start the process of DNA replication

(c) DNA-dependent DNA polymerases catalyse polymerisation only in one direction, i.e.
5'→ 3'. It creates additional complications at the replicating fork. Consequently, on one
strand (template 3' →5'), the replication is continuous. This is known as leading strand,
while on the other strand (template 5'→ 3'), it is discontinuous. This is known as lagging
strand.

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 The discontinuously synthesised fragments on lagging strand are called Okazaki fragments and
these are later joined by DNA ligase.
(x) Replication does not initiate randomly at any place in DNA. So, there is a definite region in
E. coli DNA, where the replication originates. This region is termed as origin of replication
and they are provided by [Link] help in propagation of a piece of DNA, during
recombinant DNA procedures.
(xi) The failure in cell division after DNA replication results in polyploidy.

Transcription
➢ It is the process of copying genetic information from one strand of the DNA into RNA. The
principle of complementarity governs the process of transcription, except the adenosine now
forms base pair with uracil instead of thymine.
(i) In transcription, only a segment of DNA is duplicated and only one of the strands is copied into
RNA. Both the strands are not copied because

(a) If both the strands code for RNA, two different RNA molecules and two different proteins
would be formed, hence complicating the genetic information transfer machinery.

(b) Since, two RNA produced would be complementary to each other, they would form a double-
stranded RNA without translation, making the process of transcription futile.
(ii) A transcription unit in DNA is defined by three regions in the DNA, which are
as follows
a. A promoter
b. The structural gene
c. A terminator
(iii) The two strands of DNA have opposite polarity and the DNA-dependent RNA
polymerase also catalyses the polymerisation in only one direction, that is 5'→ 3' direction.

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 (iv) The strand that has the polarity (3'→ 5′) acts as a template and is referred to as
template strand. The other strand, which has the polarity (5'→ 3′) and the sequence same as
RNA (T at the place of U) is displaced during transcription. This strand is called as coding
strand.

(v) The promoter and terminator flank the structural gene in a transcription unit.
(vi) The promoter is located towards 5' end (upstream) of the structural gene.
(vii) It is the DNA sequence that provides binding site for RNA polymerase and also the
presence of promoter defines the template and coding strands. By switching its position with
terminator, the definition of coding and template strands could be reversed.
(viii) The terminator is located towards 3' end (downstream) of the coding strand and it
usually defines the end of the process of transcription.
(ix) There are additional regulatory sequences that may be present further upstream or
downstream to the promoter.
Transcription Unit and the Gene
➢ A gene can be defined as the functional unit of inheritance. Genes are located on the
chromosomes.
➢ A cistron is a segment of DNA that codes for a polypeptide.
➢ The structural gene in a transcription unit could be monocistronic (mostly in eukaryotes) or
polycistronic (mostly in bacteria or prokaryotes).
➢ In monocistronic gene, only one cistron is present in one transcription unit, while in polycistronic
gene, many cistrones are present. In eukaryotes, monocistronic gene is splitted.
➢ The coding sequences or expressed sequences are defined as exons. Exons appear in mature
or processed RNA. The exons are interrupted by introns, i.e. the for non-coding sequences.
➢ Introns or intervening sequences do not appear in mature or processed RNA.
➢ Sometimes, the regulatory sequences are loosely defined as regulatory genes, even though these
sequences do not code for any RNA or protein.

Transcription in Prokaryotes
It occurs in the following steps
➢ A single DNA-dependent RNA polymerase catalyses the transcription of all the three types of
RNA in bacteria.
➢ RNA polymerase binds to promotor and initiates transcription (initiation).

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 ➢ It uses nucleoside triphosphates as substrate' and polymerises in a template depended fashion
following the rule of complementarity.
➢ It also facilitates opening of the helix and continues elongation.
➢ Once the polymerase reaches the terminator region, the nascent RNA and RNA polymerase falls
off. This results in termination of transcription.
➢ RNA polymerase is only capable of catalysing the process of elongation. It associates transiently
with initiation- factor (σ) and terminator factor (ρ), to initiate and terminate the process of
transcription, respectively. In other words, RNA polymerase can identify promoter only when σ
factor is attached on the promoter. Similarly it terminates the process of transcription when a p
factor is attached on the terminator. Thus, catalysing all the three steps,
➢ In bacteria, since the mRNA does not require any processing to become active and also since
transcription and translation take place in the same compartment, many times the
translation can begin much before the mRNA is fully transcribed. As a result, transcription and
translation can be coupled in bacteria.

Transcription in Eukaryotes
Eukaryotes have additional complexities than prokaryotes as follows
➢ There are at least three RNA polymerases in the nucleus other than the RNA polymerase. The
RNA polymerase-I transcribes rRNAS (28S, 18S and 5.8S).RNA polymerase-III is
responsible for the transcription of tRNA, 5srRNA and snRNAs (small nuclear RNAs).
RNA polymerase-II transcribes precursor of mRNA, the heterogenous nuclear RNA
(hnRNA).
➢ Another complexity is that, the primary transcripts contain both the exons and the introns
and are non-functional.
➢ Hence, it is subjected to a process called splicing. In this process, introns are removed and exons
are joined in a definite order.
➢ hnRNA undergoes additional processing called capping and tailing. In capping, an unusual
nucleotide (methyl guanosine triphosphate) is added to the 5' end of hnRNA.
➢ In tailing, adenylate residues (200-300) are added at 3' end in a template. Capping and tailing
protect the mRNA from degradation by the activity of digestive enzymes present in the
cytoplasm.
➢ The fully processed hnRNA, is called mRNA, that is transported out of the nucleus for
translation process.

Significance of these complexities are

➢ The split gene arrangements represent an ancient feature of genome.
➢ The presence of introns is reminescent of antiquity.
The process of splicing represents the dominance of RNA world

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 Genetic Code
➢ Genetic code is a sequence of three nucleotides present on mRNA, which codes for a specific
amino acid during translation.
➢ It is the relationship between the sequences of nucleotides on mRNA and the sequences of
amino acids in the polypeptide chain.
Deciphering the Code
➢ George Gamow, a physicist suggested that the genetic code should be made up of three
nucleotides. He stated that since there are only four bases and if they have to code for 20 amino
acids, the code should constitute a combination of bases. But, a permutation combination of 43(4
× 4 × 4) would generate 64 codons, i.e. many more codons than required
➢ Har Gobind Khorana could synthesise RNA molecules with defined combinations of bases
(homopolymers and copolymers).
➢ Marshall Nirenberg made cell-free system for protein synthesis and finally the code was
deciphered.
➢ Severo Ochoa enzyme (polynucleotide phosphorylase) was also helpful in polymerising RNA
with defined sequences in a template independent manner (enzyme synthesis of RNA).
➢ All of these investigations, finally helped to make a checker-board for genetic code as given
follow:

Salient Features of Genetic Code

These are as follows
(i) A codon is a triplet. Out of 64 codons, 61 codons code for amino acids and 3 codons do not
code for any amino acids. Hence, they function as stop codons [UAA, UAG and UGA].
(ii) One codon codes for only one amino acid, hence, it is unambiguous and specific.
For example, CCG codes for proline, it will not code for any other amino acid.
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 (iii) Some amino acids are coded by more than one codon, hence the code is degenerate.
(iv) The codon is read in mRNA in a contiguous fashion. There are no punctuations.
(v) The code is nearly universal. For example, from bacteria to human, UUU would code for
phenylalanine (Phe). Some exceptions are found in mitochondrial codons and in some
protozoans.
(vi) AUG has dual functions. It codes for methionine (met) and also acts as initiator
codon.

Mutations
The sudden inheritable change in the genetic material is defined as mutation.
It can be of following types
(i) Point mutation occurs due to change in single base pair. For example, change of single base
pair at the sixth position in the gene for ẞ-globin chain of haemoglobin results in the change of
amino acid residue glutamate to valine. It results in sickle-cell anaemia.
(ii) Frameshift mutation occurs where addition/insertion or deletion of one or two bases
changes the reading frame from the site of mutation, resulting in a protein with a different set of
amino acids. Insertion or deletion of three of its multiples of bases do not alter the reading frame,
but due to this, one or more amino acids coded in the protein get translated.
(iii) Silent mutation occurs when a base change in a codon does not alter the amino acid to be
coded. This forms the genetic basis of proof that codon is a triplet and it is read in a contiguous
manner.

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 Translation
➢ It is the process of polymerisation of amino acids to form a polypeptide.
(i) The order and sequence of amino acids are defined by the sequence of bases in the mRNA.
(ii) The amino acids are joined by a bond, which is known as a peptide bond. This process
requires energy.
(iii) The process of translation requires the following components- mRNA, ribosome, amino
acids as raw material, ATP, adaptor molecule-tRNA and aminoacyl tRNA synthetase
enzyme for charging of tRNA.

tRNA-The Adapter Molecule

➢ Francis Crick postulated the presence of an. adapter molecule that would read the code and bind
to specific amino acids. This molecule is called tRNA. It may also be called as sRNA, i.e.
soluble RNA as it is soluble in water. tRNA was known before the genetic code was postulated
but its role as an adapter molecule was assigned much later.
➢ Clover leaf model shows tRNA has an anticodon loop that has bases complementary to the code.
It also has an amino acid acceptor end, which helps it to bind to amino acids. It also possesses an
enzyme site and a ribosome site.

➢ tRNAs are specific for each amino acid For initiation, there is specific tRNA called initiator
tRNA. There is no tRNA for stop codons.
➢ The secondary structure of tRNA looks like a clover-leaf. In actual structure, it is a compact
molecule that appears like inverted L.

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 Phases of Translation
(i) Activation of amino acids occurs in the presence of ATP and link to their cognate tRNA, in
the presence of aminoacyl tRNA synthase enzyme. It is known as charging of tRNA or
aminoacylation of tRNA. If two such charged tRNAs are brought closer, the formation of
peptide bond between them would occur energetically in the presence of a catalyst.

(ii) Initiation of polypeptide synthesis occurs in ribosomes which is known as the cellular
factory for protein synthesis.
❖ Ribosome consists of structural RNAs and about 80 different proteins.
❖ In its inactive state, it exists as two separated subunits, i.e. a large and a small subunit.
❖ When the small subunit encounters an mRNA, the process of translation of the mRNA to protein
begins.
❖ There are two sites in the large subunit, i.e. the P-site and A-site for subsequent binding of
amino acids. Thus, these are close enough to each other for the formation of a peptide bond.
❖ The small subunit (with the tRNA) attaches to the large subunit in such a way that the initiation
codon (AUG) comes to the P-site.
❖ An mRNA also has some additional sequences that are not translated. These are referred to as
Untranslated Regions (UTR). They are present at both 5' end (before start codon) and at 3' end
(after stop codon) for efficient translation process.
(iii) Elongation of polypeptide chain occurs when a second tRNA charged with an appropriate
amino acid binds to the A-site of the ribosome.
❖ A peptide bond (CO-NH) forms between the carboxyl group of methionine and the amino
group of the second amino acid. The reaction is catalysed by the enzyme peptidyl transferase.

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 ❖ The complexes consisting of an amino acid linked to tRNA, sequentially bind to the appropriate
codon in mRNA by forming complementary base pairs with the tRNA anticodon.
❖ The ribosome moves from codon to codon along the mRNA in 5'→ 3' [Link] acids are
added one by one,
(iv) Termination of polypeptide synthesis occur when a release factor binds to the stop codon.
As a result, the polypeptide synthesis or elongation process stops, releasing the complete
polypeptide from the ribosome.

Regulation of Gene Expression

It occurs at various levels and results in the formation of a polypeptide.
i. In prokaryotes, gene expression is regulated by the rate of initiation of transcription.
ii. In eukaryotes, regulation is achieved at four levels
a. Transcriptional level (formation of primary transcript).
b. Processing levels (regulation of splicing).
c. Transport of mRNA from nucleus to the cytoplasm.
d. Translational level.
iii. Genes in a cell are expressed to perform a particular function or a set of functions.
iv. The metabolic, physiological or environmental conditions regulate expression of genes.
v. The development and differentiation of embryo into adult organisms are also a result of
coordinated regulation of expression of several sets of genes.
vi. In a transcriptional unit, the activity of RNA polymerase at a given promoter is in turn regulated
by the interaction with accessory proteins.
vii. In many cases, the accessibility to promoter regions of prokaryotic DNA is regulated by the
interaction of proteins with sequences, termed as operators.
viii. The sequences of the operator bind a repressor protein. Each operon has its specific
operator and specific repressor. For example, lac operon interacts with lac repressor only.
F Jacob and J Monod were the first to describe a transcriptionally regulated system.
i. An operon is a unit of prokaryotic gene expression, which includes sequentially regulated
(structural) genes and control elements recognised by the regulatory gene product. In other
words, when many genes are regulated by a single promoter then this arrangement of genes is
known as operon.
ii. The various components of an operon are
a) Structural genes Fragments of DNA, which transcribe mRNA for polypeptide synthesis.
b) Promoter gene Sequence of DNA, where RNA polymerase binds and initiates transcription.
c) Operator Sequence of DNA adjacent to promoter, where specific repressor protein binds.
d) Regulator gene codes for the repressor protein that binds to the operator and suppresses its
activity, hence transcription does not occur. Also, represented as 'i' gene.
e) Inducer prevents the repressor from binding to the operator. Due to this, transcription is
switched on.
➢ It may be a metabolite, hormone, etc. In lac operon, which is related to lactose metabolism, a
polycistronic structural gene is regulated by a common promoter and regulatory genes.
iii. The lac operon consists of one regulatory gene (i gene) and three structural genes (z, y and a).

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 a) i-for repressor of lac operon z-for beta-galactosidase (ẞ-gal) that catalyses the hydrolysis of
lactose into galactose and glucose. y-for permease that increases the permeability of the cell.
a-for transacetylase.
b) All the three gene products in lac operon are required for the metabolism of lactose.
c) In case of bacteria, when glucose is present in the nutrition media, lac operon is closed.
iv. But when glucose is replaced by lactose, then it needs ẞ-galactosidase enzyme to break lactose
into glucose and galactose, hence, lac operon is activated.
v. Lactose is a substrate for enzyme B-galactosidase and it regulates switching on and off of the
operon,. hence termed as inducer.
vi. The lactose induces operon in the following ways
a) When glucose is present in the medium, repressor of the operon is synthesised from the i gene.
b) Repressor protein binds to the operator region of the operon, so
RNA polymerase cannot bind on promoter and transcription does not occur. Hence, B-
galactosidase is not formed.
c) In the presence of an inducer, such as lactose or allolactose, the repressor is inactivated by
interaction with the inducer. So, now inactivated repressor cannot bind on operator.
vii. This allows RNA polymerase access to the promoter and transcription proceeds. It means all the
genes are transcribed and all three enzymes are formed.
d) Regulation of lac operon by a repressor is referred to as negative regulation.

Human Genome Project (HGP)

➢ It is a 13 year project coordinated by the US Department of Energy and the National Institute
of Health. It was launched in the year 1990 and completed in 2003. It was closely associated
with rapid development of a new area in biology called bioinformatics. HGP was called as a
megaproject because
(i) total cost of the project was approximately 9 billion US dollars.

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 (ii) to store the information, it will require 3300 books of 1000 pages each, if 1000 letters are
written on each page.
(iii) it needs high speed computational devices to store, retrieve and analyse the information.

The important goals of HGP are to

a) identify all the approximately 20000-25000 genes in human DNA.
b) determine the sequences of the 3 billion chemical base pairs that make up human ..DNA.
c) store this information in databases.
d) improve tools for data analysis.
e) transfer related technologies to other sectors, such as industries.
f) address the Ethical, Legal and Social Issues (ELSI) that may arise from the project.

Methodologies of HGP
These are focussed on two main lines, i.e. expressed sequence tags and sequence annotation
(i) Expressed Sequence Tags (ESTs) method is based on identifying all the genes that are
expressed as RNA.
(ii) Sequence annotation is the approach of simply sequencing the whole set of genome that
contains all the coding and non-coding sequences and later assigning different regions in the
sequence with functions.
(a) For sequencing, the total DNA from cell is isolated and converted into relatively smaller size as
fragments.
(b) DNA fragments are cloned in suitable host using specialised vectors, such as Bacterial Artificial
Chromosome (BAC) and Yeast Artificial Chromosome (YAC).
(c) Fragments of DNA are then sequenced by automated DNA sequences, which work on the
principle developed by F Sanger.
(d) These sequences are arranged accordingly on the basis of overlapping regions on DNA
fragments.
(e) The alignments of these sequences based on computer programmes are developed.
(f) At last, the genetic and physical maps of the genome are constructed by collecting information
about certain repetitive DNA sequences and DNA polymorphism.

Salient Features of Human Genome

These are as follows
(i) The human genome contains 3164.7 million nucleotide bases.
(ii) The average gene consists of 3000 bases, but sizes vary greatly, with the largest known
human gene being dystrophin of 2.4 million bases.
(iii) The total number of genes is estimated at 30000, much lower than previous estimates of
80000-140000 genes. Almost all (99.9%) nucleotide bases are exactly the same in all
people.
(iv) The functions of over 50% of discovered genes are unknown.
(v) Less than 2% of the genome codes for proteins.
(vi) Repeated sequences make up very large portion of the human genome.

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 ➢ These sequences are stretches of DNA sequences that are repeated many times, sometimes
hundred to thousand times. They are thought to have no direct coding functions, but shed light on
chromosome structure, dynamics and evolution.
(vii) Chromosome 1 has most genes (2968) and the Y has the fewest (231).
(viii) Scientists have identified about 1.4 million locations, where single base DNA differences,
Single Nucleotide Polymorphisms (SNPs) occur in humans.

Applications of HGP
(i) Knowledge from DNA sequences will define research leading to our knowledge of biological
systems.
(ii) It will enable a radically new approach to biological research.
(iii) It can be used to study genes in a genome.
(iv) It can create new ways to diagnose, treat and prevent the disorders that affect humans.
(v) Many non-human model organisms such as C. elegans, Drosophila, rice, Arabidopsis have
also been sequenced.

DNA Fingerprinting
➢ It is a quick way to compare the DNA sequences of any two individuals.
(i) Dr. Alec Jeffreys developed the technique of DNA fingerprinting in an attempt to identify
DNA marker for the inherited diseases.
(ii) DNA fingerprinting involves identifying differences in some specific regions in DNA
sequence called as repetitive DNA, i.e. a small stretch of DNA is repeated many times.
(iii) The repetitive DNAs are separated from bulk genomic DNA as different peaks during density
gradient centrifugation. The bulk DNA forms a major peak and the other small peaks are
referred to as satellite DNA.
(iv) Satellite DNA can be classified as microsatellites, minisatellites, etc., depending on base
composition, the length of segment and number of repetitive units.
(v) These sequences show high degree of polymorphism and form the basis of DNA
fingerprinting.
(vi) Since DNA from every tissue (as blood, hair, etc.) from an individual differs, they become
very useful tool in forensic applications.
(vii) As the polymorphisms are inheritable, DNA fingerprinting is the basis of paternity testing as
well.
(viii) Polymorphism (variation at genetic level) arises due to mutations. If an inheritable mutation
is observed in a population of high frequency, it is called DNA polymorphism.
(ix) The mutations keep on accumulating generation after generation and form one of the basis of
variability/polymorphism.
(x) There is a variety of different types polymorphism ranging from single nucleotide change to
very large scale changes.

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 Methodology of DNA Fingerprinting
➢ This technique involves Southern blot hybridisation, using radiolabelled VNTR as a probe.

The methodology includes

(i) DNA is isolated and digested by the restriction endonucleases.
(ii) DNA fragments are separated by electrophoresis.
(iii) Separated DNA fragments are transferred to synthetic membranes like nitrocellulose or
nylon.
(iv) Hybridisation using labelled VNTR probe.

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 (v) Hybridised DNA fragments are detected by autoradiography.

The sensitivity has been increased by the use of Polymerase Chain Reaction (PCR). Variable
Number of Tandem Repeats (VNTRS) belongs to a class of satellite DNA called minisatellite.
(i) The chromosome number repeats show very high degree of polymorphism. Due to this, the
size of VNTR varies in size from 0.1-20 kb.
(ii) This DNA differs in individual to individual except in case of monozygotic (identical)
twins.
(iii) Consequently, after hybridisation with VNTR probe, the autoradiogram gives many bands of
different sizes. These bands give characteristic pattern of an individual DNA.

Applications of DNA Fingerprinting

(i) It is useful as identification tool in forensic applications.
(ii) It is the basis of paternity testing in case of disputes.
(iii) It is used in determining population and genetic diversities and also in evolutionary biology.

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